Docking Analysis of 17 Selected Artoindonesianins as Human Neutrophil Elastase, Matrix Metalloproteinase 9 and G9A like Protein Lysine Methyl Transferase Modulating agents.

INTRODUCTION

The genus Artocarpus comprises of fifty species, which are distributed in tropical and subtropical regions of Asia (Rao et al., 2010). “Artocarpus” is derived from the Greek word ‘artos’ and ‘karpos’ which means ‘bread’ and ‘fruit’ respectively. Bread fruits are rich source of carbohydrates and minerals consuming it helps to overcome malnutrition (Buddhisuharto et al., 2021). Especially bread fruit seeds are rich in i) Manganese (Mn), ii) Magnesium (Mg), iii) Potassium (K), iv) Calcium (Ca), v) Iron (Fe) and lectins which meet up the nutritional requirements for the poor people (Khan et al., 2021). Artocarpus species are abundant in phenols, flavonoids, stilbenoids, arylbenzofurons and lectin (Baliga et al., 2011). Preclinical studies have reported that it possesses antifungal, anti-neoplastic, hypoglycemic and wound healing activities.

The Artocarpus genus are an important natural source of phytochemicals like i) artocarpin-A, ii) artocarpin-B, iii) morin, iv) dihydromorin, v) cynomacurin, vi) artocarpin, vii) cyloartocarpin, viii) artocarpesin, ix) oxydihydroartocarpesin, x) artocarpetin, xi) norartocarpetin, xii) cycloartinone, xiii) ursolic acid, xiv) β-sitosterol, xv) betulinic acid, xvi) artocarpanone, and xvii) heterophylol (Somashekhar et al., 2013). Apart from these several prenylated flavonoids have been reported from various Artocarpus species and each phytochemical is unique to specific Artocarpus species. For instance, i) both Artoindonesianin A and Artoindonesianin B have been isolated from the roots of Artocarpus champeden (Hakim et al., 1999); ii) Artoindonesianin P was obtained from A. lanceifolius (Hakim et al., 2002); while Artoindonesianin L has been reported from A. rotunda (Suhartati et al., 2001); similarly Artoindonesianin C has been isolated from A. elasticus (Fajriah et al., 2022). Further, two more (Artoindonesianin O and N) have been reported from A. gomezianus (Hakim et al., 2002). Four Artoindonesianins (namely Artoindonesianins Q. R, S and T) have been reported from A. champeden (Syah et al., 2002). Similarly, two more Artoindonesianins (Artoindonesianins U and V) have been reported from A. champeden (Syah et al., 2004). Artoindonesianin E1 (oxepinoflavone) has been reported from A. elasticus (Musthapa et al., 2009); another Artoindonesianin (Artoindonesianin J) has been reported from A. bracteata (Ersam et al., 2002). Further two more Artoindonesianins (Artoindonesianins X and Y) have been reported from A. fretessii (Soekamto et al., 2003).

The earlier studies engaged us to carry out the current investigation on 17 chosen Artoindonesianins (Artoindonesianin A, B, B1, C, E1, J, L. N, O, P, Q, R, S, T, U, V and Y) which were aimed to investigate on the docking analysis of human neutrophil elastase (hNE), human matrix metalloproteinase 9 (hMMP 9) and human G9A like protein lysine methyl transferase (hPKMT) by using the swissdock method, which aids in developing anti-inflammatory/anti-cancer agents for managing inflammation and cancer related disorders.

MATERIALS AND METHODS

Ligand preparation

The chemical structures of 17 selected Artoindonesianins ligands were selected for the present investigation based on previous reports, that includes 1) Artoindonesianin A (CID 10603133), 2) Artoindonesianin B (CID 10096171), 3) Artoindonesianin B1 (CID 71307312), 4) Artoindonesianin C (CID 10552003), 5) Artoindonesianin E1 (CID 70678703), 6) Artoindonesianin J (CID 15549721), 7) Artoindonesianin L (CID 163184360), 8) Artoindonesianin N (CID 71626927), 9) Artoindonesianin O (CID 15700061), 10) Artoindonesianin P (CID 10316935), 11) Artoindonesianin Q (CID 5320428), 12) Artoindonesianin R (CID 5320437), 13) Artoindonesianin S (CID 5320444), 14) Artoindonesianin T (CID 5320451), 15) Artoindonesianin U (CID 10030972), 16) Artoindonesianin V (CID 10053761), 17) Artoindonesianin Y (CID 641711), 18) Sivelestat - hNE reference compound1 (CID 107706), 19) Artoindonesianin F - hNE reference compound2, 20) Caffeic acid – hMMP 9 reference compound1 (CID 689043), 21) Genistein – hMMP 9 reference compound2 (CID 5280961), 22) BIX-01294 – hPKMT reference compound1 (CID 124219522) and 23) MS8511 – hPKMT reference compound2 (CID 164512403) were downloaded from PubChem compound database. These 17 selected Artoindonesianins and 6 reference compounds structures were drawn and prepared by using ChemDraw 2D and 3D software tools (Mohan et al., 2022). Thus, these prepared three-dimensional structures were used for further studies (swissdock).

Preparation of target enzymes

The three-dimensional [3-D] structures of human neutrophil elastase [hNE] (PDB^◘◘ ID: 1H1B with a resolution of 2.00 Aᵒ), human matrix metalloproteinase 9 [hMMP 9] (PDB^◘◘ ID: 4H1Q with a resolution of 1.59 Aᵒ) and human G9a-like protein lysine methyltransferase (hPKMT) (PDB^◘◘ ID: 3FPD with a resolution of 2.40 Aᵒ) was downloaded from ^◘◘Protein Data Bank (PDB) databases respectively. ‘A’ chain of all three enzymes (hNE, hMMP 9 and hPKMT) were prepared independently by removing other chains, ligands, and even the crystallographically observed “water” (H₂O) molecules by using UCSF Chimera software tool (Mohan et al., 2022).

Toxicity analysis

Toxicity analysis was performed for 17 chosen Artoindonesianins through the ‘pkCSM’ free online server (Prakash et al., 2023).

Docking study

A docking studies was performed for 17 chosen Artoindonesianins with three target proteins (hNE, hMMP 9 and hPKMT) using the Swissdock free online server (Prakash et al., 2023). Finally, PLIP (Protein-Ligand Interaction Profiler) free web server was utilized to analysis the binding site of best-docked pose for each ligand (Srinivasan et al., 2023).

Statistical analysis

Docking protocol was validated using sivelestat (hNE), caffeic acid (hMMP 9) and BIX-01294 (hPKMT) as reference compounds. And the Root Mean Square Deviation [RMSD**] analysis of all the docked complexes (17 target ligands) was separately compared with that of (reference compounds) docked complex for each chosen target enzymes by utilizing the ‘align’ command in “PyMOL” software (Ramsbottom et al., 2018, Narayanaswamy et al. 2024).

In present investigation, no experimental animals (or) human subjects were used, thus ethical approval was not needed.

RESULTS

In the present study, Table 1 represents the toxicity analysis of 17 chosen Artoindonesianins (ligands), in which two ligands (Artoindonesianin C and P) were predicted to possess hepatotoxicity nature.

The current molecular docking analysis showed that Artoindonesianin U has the Highest Binding Energy (HBE) (-8.03 kcal/mol) with the Human Neutrophil Elastase (hNE). In contrast, Artoindonesianin A had the minimum binding energy (MBE) (-6.29 kcal/mol) with the human Neutrophil Elastase (hNE) (as shown in Table 2).

Eleven ligands (Artoindonesianin B1, C, E1, J, N, Q, R, S, T, V and Y) have shown interactions with Ser 195 amino acid residue of human Neutrophil Elastase (hNE). Similarly, eight ligands (Artoindonesianin B1, C, L, N, O, R, V and Y) have shown interaction with His 57 amino acid residue of human Neutrophil Elastase (hNE). However, one ligand (Artoindonesianin A) does not show any hydrogen bond interaction with the amino acid residue of human neutrophil elastase (hNE), as shown in Figure 1a.

Click to enlarge

The present molecular docking results showed that Artoindonesianin L has the Maximum Binding Energy (MBE) (-9.14 kcal/mol) with the Human Matrix Metalloproteinase (hMMP 9) enzyme. On the other hand, Artoindonesianin A had the least binding energy (LBE) (-6.57 kcal/mol) with the human Matrix Metalloproteinase (hMMP 9) enzyme (as shown in Table 3).

Seven ligands (Artoindonesianin J, L, P, Q, S, U and Y) have shown interactions with Gln 227 amino acid residue of human Matrix Metalloproteinase (hMMP 9). Similarly, four ligands (Artoindonesianin C, P, S and V) have shown interaction with Tyr 248 amino acid residue of human Matrix Metalloproteinase (hMMP 9). However, three ligands (Artoindonesianin B [as shown in Figure b, E1 and J) do not show any hydrogen bond interaction with the amino acid residue of Human Matrix Metalloproteinase (hMMP 9).

The present molecular docking analysis showed that Artoindonesianin U has the Highest Binding Energy (HBE) (-9.28 kcal/mol) with the human G9a-like protein lysine methyltransferase (hPKMT). In contrast, Artoindonesianin V had the Minimum Binding Energy (MBE) (-6.61 kcal/mol) with the human G9a-like Protein Lysine Methyltransferase (hPKMT) (as shown in Table 4).

Four ligands (Artoindonesianin B1, N, T and U) have shown interactions with Trp 1107 amino acid residue of human G9a-like protein lysine Methyltransferase (hPKMT). Similarly, four ligands (Artoindonesianin B1, P, R and T) have shown interaction with Arg 1226 amino acid residue of human G9a-like protein lysine Methyltransferase (hPKMT). However, one ligand (Artoindonesianin L) does not show any hydrogen bond interaction with the amino acid residue of human G9a-like protein lysine Methyltransferase (hPKMT) as shown in Figure c.

DISCUSSION

Artocarpus heterophyllus (AH) is one among the nine Artocarpus species reported from India, which belongs to mulberry (Moracaea) family (Rao et al., 2010). A. heterophyllus (AH) is known by various regional names for instance i) Jackfruit (English), ii) Palaa (Tamil), iii) Phanas (Gujarati and Marathi) and iv) Chakka (Malayalam). A. heterophyllus (AH) is native to Western Ghats of India, Malaysia and also found in Central and Eastern Africa (Prakash et al., 2009). Traditionally A. heterophyllus (AH) is used to treat diabetes, diarrhea, inflammation, malarial fever, and tape worm infections. Moreover, Artoindonesianin F has been isolated from A. heterophyllus and reported to inhibit tyrosinase activity (Rao et al., 2010). Furthermore, Artoindonesianin F has been reported to inhibit human Neutrophil Elastase (hNE) using molecular docking method (Narayanaswamy et al., 2013). Thus, in the present study, Artoindonesianin F has been chosen as one of reference compound for human neutrophil elastase (hNE).

Prior to docking, toxicity analysis was carried out in the present study where two (Artoindonesianin C and P) ligands were predicted to possess hepatotoxicity nature. Moreover, Artoindonesianin P has been shown to possess cytotoxicity against P388 (murine leukemia) cells (Hakim et al., 2002). Furthermore, Artoindonesianin U and V have been reported to possess cytotoxicity against P388 (murine leukemia) cells (Syah et al., 2004). However, leaf ethyl acetate fractions of A. altilis, A. champeden and A. heterophyllus have demonstrated to possess hepatoprotective effect against CCl4 induced liver injury in rat model (Fitrya et al., 2024).

The chosen ligands (Artoindonesianins) have shown interaction with His57, Asn61, Arg147, Gly193 and Ser195 Amino Acid Residues (AAR) of human neutrophil elastase (hNE) enzyme. This finding showed good agreement with previous reports (Mohan et al., 2022, Ragavan et al., 2026).

Similarly selected ligands (Artoindonesianins) have exhibited interaction with Leu188, Ala189, Ala191, Gln227 and Tyr248 Amino Acid Residues (AAR) of human matrix metalloproteinase 9 (hMMP 9) enzyme. This result showed excellent correlation with earlier reports (Ragavan et al., 2020, Radhakrishnan et al., 2023, Ragavan et al., 2026).

In the present study, one ligand (Artoindonesianin S) has shown interaction with Asp1145 and Arg1214 Amino Acid Residues (AARs) of human G9a-like protein lysine methyltransferase (hPKMT). This finding was on par with the previous reports (Zang et al., 2017, Rahman et al., 2021).

CONCLUSION

In the current investigation, the 17 chosen Artoindonesianins of Artocarpus species have shown the potential to dock with three targeted human enzymes (hNE, hMMP 9 and hPKMT). However, one ligand (Artoindonesianin A) does not show any hydrogen bond interaction with the amino acid residue of Human Neutrophil Elastase (hNE). Moreover, three ligands (Artoindonesianin B, E1 and J) do not show any hydrogen bond interaction with the amino acid residue of Human Matrix Metalloproteinase (hMMP 9). Furthermore, one ligand (Artoindonesianin L) does not show any hydrogen bond interaction with the amino acid residue of human G9a-like protein Lysine Methyltransferase (hPKMT). Thus, the present finding give new insight about the 17 chosen Artoindonesianins of Artocarpus species as potent modulating agents of hNE, hMMP 9 and hPKMT which will help in managing cancer, inflammation, photo-aging, and wounds.